The present invention relates to a novel chemokine. More specifically, isolated nucleic acid molecules are provided encoding a human Chemokine Alpha-4, hereinafter referred to as xe2x80x9cCKxcex1-4xe2x80x9d. CKxcex1-4 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of CKxcex1-4 activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.
The ability to control the migration and xe2x80x9ctraffickingxe2x80x9d of various cell types is controlled by a subset of factors, or proteins, among which chemokines are an example. Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related chemotactic cytokines. These molecules are 8-12 kDa in size. In general, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamnily, the first two cysteines are separated by one amino acid and hence are referred to as the xe2x80x9cCxe2x80x94Xxe2x80x94Cxe2x80x9d subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the xe2x80x9cCxe2x80x94Cxe2x80x9d subfamily. Thus far, over a dozen different members of this family have been identified in humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, interleukin-8 (L-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
Members of the xe2x80x9cCxe2x80x94Cxe2x80x9d subfamily exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophages which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation. However, members of one subfamily may exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches.
While members of the Cxe2x80x94C subfamily act predominantly on mononuclear cells and members of the Cxe2x80x94Xxe2x80x94C subfamily act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one subfamily show characteristics of the other.
The polypeptide of the present invention has the conserved cysteine residues of the xe2x80x9cCxe2x80x94Xxe2x80x94Cxe2x80x9d region, and have amino acid sequence homology to known chemokines.
Clearly, there is a need for factors that regulate the migration of distinct cell types and their roles in dysfunction and disease. There is a need, therefore, for identification and characterization of such factors that regulate the migration of cells, particularly cells of the immune system, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CKxcex1-4 polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97692 on Aug. 27, 1996. The nucleotide sequence determined by sequencing the deposited CKxcex1-4 clone, which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 94 amino acid residues, including an initiation codon at positions 66-68, with a leader sequence of about 18 amino acid residues, and a predicted molecular weight of about 10 kDa. The amino acid sequence of the predicted mature CKxcex1-4 protein is shown in FIG. 1, amino acid residues 19-94 (also residues 19-94 in SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CKxcex1-4 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the complete CKxcex1-4 polypeptide having the amino acid sequence shown in FIG. 1 excepting the N-terminal methionine (i.e., residues 2 to 94 in SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted mature CKxcex1-4 polypeptide having the amino acid sequence at positions 19-94 in FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequence encoding the CKxcex1-4 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97692; (e) a nucleotide sequence encoding the mature CKxcex1-4 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97692; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e) or (f), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a CKxcex1-4 polypeptide having an amino acid sequence in (a), (b), (c), (d) or (e), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of CKxcex1-4 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated CKxcex1-4 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the CKxcex1-4 polypeptide having the complete 94 amino acid sequence, including the leader sequence shown in FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of the CKxcex1-4 polypeptide shown in FIG. 1 excepting the N-terminal methionine (i.e., residues 2 to 94 shown in SEQ ID NO:2) (c) the amino acid sequence of the predicted mature CKxcex1-4 polypeptide (without the leader) having the amino acid sequence at positions 19-94 in FIG. 1 (SEQ ID NO:2); (d) the amino acid sequence of the CKxcex1-4 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97692; and (e) the amino acid sequence of the mature CKxcex1-4 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.97692. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c), (d) or (e) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a CKxcex1-4 polypeptide having an amino acid sequence described in (a), (b), (c), (d) or (e), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a CKxcex1-4 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a CKxcex1-4 polypeptide having an amino acid sequence described in (a), (b), (c), (d) or (e) above.
The invention further provides methods for isolating antibodies that bind specifically to a CKxcex1-4 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the CKxcex1-4, which involves contacting cells which express the CKxcex1-4 with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in the absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on CKxcex1-4 binding to the CKxcex1-4 receptor. In particular, the method involves contacting the CKxcex1-4 receptor with a CKxcex1-4 polypeptide and a candidate compound and determining whether CKxcex1-4 polypeptide binding to the CKxcex1-4 receptor is increased or decreased due to the presence of the candidate compound.
The present inventor has discovered that CKxcex1-4 is expressed in g-interferon induced epithelial tissue. For a number of immune system-related disorders, significantly higher or lower levels of CKxcex1-4 gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d CKxcex1-4 gene expression level, i.e., the CKxcex1-4 expression level in healthy tissue from an individual not having the immune system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of immune system disorders, which involves: (a) assaying the CKxcex1-4 gene expression level in cells or body fluid of an individual; (b) comparing the CKxcex1-4 gene expression level with a standard CKxcex1-4 gene expression level, whereby an increase or decrease in the assayed CKxcex1-4 gene expression level compared to the standard expression level is indicative of an immune system disorder.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of CKxcex1-4 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated CKxcex1-4 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of CKxcex1-4 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an CKxcex1-4 antagonist. Preferred antagonists for use in the present invention are CKxcex1-4-specific antibodies and CKxcex1-4 ELR mutants.